Both T cell and B cell immune responses to HCV are being investigated. Previously we developed and characterized human and mouse monoclonal antibodies to the HCV structural genes; core, E1 and E2 using recombinant baculovirus expressed antigens. We have shown that the antibodies to E1 and E2 can bind to native virion and antibody to core can bind to virion stripped of its envelope by detergent. T cell studies are continuing in an effort to identify both cytotoxic T cell and helper T cell epitopes. We are studying the proliferative and CTL responses of patients with chronic HCV infections to peptide antigens and antigens expressed by recombinant DNA systems. We previously identified a CTL epitope in the core region that is recognized by both mice and humans. Using predicted HLA A2 motifs from the HCV polyprotein, we identified five additional epitopes, 3 in the core protein, 1 in NS4B and 1 in NS5B. We found that HLA A2 patients with chronic HCV infections react to at least one of these epitopes in CTL assays but that 2 patients who had recovered from HCV did not react. We have also developed a new approach to identifying CTL epitopes that is not dependent on selecting for known motifs. We made proteolytic digests purified HCV NS3 protein and separated the resulting peptides by HPLC. Each fraction was used to pulse syngenetic target cells and those that were recognized in a CTL assay were sequenced. The precise epitope was then mapped by synthesizing overlapping decapeptides that covered the reactive peptide. We identified a new epitope in the NS3 protein to which patients with chronic HCV infections react. This epitope was also recognized only by patients who expressed HLA A2 even though the peptide did not contain a recognized A2 binding motif. Experimental genetic vaccines against HCV are being studied. BALB/c mice have been shown to develop antibody after injection with a plasmid that expresses HCV E2. We have extended these experiments to include the HCV core and E1 genes as well as E2. Inoculated mice exhibit both antibody and CTL responses to specific HCV antigens. In vitro correlates of immunity are being developed that will assist in evaluation of these plasmid based vaccines. Additional constructs are now being evaluated that include genes for cytokine expression as well as specific CTL epitopes from non-structural genes and non-specific helper epitopes.